A significant characteristic of the immune and inflammatory responses is the movement of leukocytes from the bloodstream into specific tissues in response to various physiological signals. For example, certain subsets of lymphocytes "home" to various secondary lymphoid tissues such as lymph nodes or Peyer's patches, and eventually return to circulation. Other leukocytes such as granulocytes and monocytes, however, do not return to circulation after transmigration from the bloodstream. Movement of leukocytes from circulation is effected by a series of receptor/counter-receptor interactions which are coordinated by various specific membrane adhesion molecules.
Extravasation of leukocytes from the bloodstream for review, see McEver, Curr. Opin. Cell Biol. 4:840-849 (1992)! is initially effected by a family of membrane glycoproteins termed selections which are either expressed constitutively or induced in response to specific cytokines. Binding of selections to their counterpart ligand brings leukocytes into close, but not static, contact with vascular endothelial cells. The "tethered" leukocyte then begins a "rolling" process along the endothelium which continues until additional molecular interactions firmly stabilize a specific cell/cell interaction. One of the molecular binding activities which results in the stable interaction is effected by a second family of surface glycoproteins called integrins which possess a higher binding affinity for their respective ligands than selectins.
The integrins are heterodimeric surface molecules comprised of an .alpha. and a .beta. subunit in non-covalent association. All integrins are transmembrane proteins with counter-receptor binding activity localized in the extracellular domain. Integrins also possess relatively short cytoplasmic regions which participate in transmembrane signaling events. Integrins are capable of interacting with other cell-bound counter-receptors and components of the extracellular matrix, as well as soluble factors. Binding of extracellular ligands leads to crosslinking and localized clustering of integrins Miyamoto, et al., Science 267:833, 1995! and formation of focal adhesions wherein the clustered integrin cytoplasmic domains associate with cytoskeletal components including, for example, actin filaments Pavalko and Otey, Proc. Soc. Exp. Biol. Med. 205:32767, 1994, and Gumbiner, Neuron 11:551, 1993!. While most investigations into integrin physiological activity have focused on identifying specific counter-receptors using immunological methodologies as discussed infra, less is known about the specific interactions of integrins with cytoplasmic components. Mutation studies, however, have indicated that the cytoplasmic sequences are required for integrin association with focal contacts and integrin dependent cell adhesion LaFlamme, et al., J. Cell. Biol. 117:437 (1992)!. Other data discussed infra support this observation.
While numerous integrins have been identified, certain subsets are unique to leukocytes, with each member of the subset having characteristic cell-specific expression and counter-receptor binding properties. Of leukocyte-specific integrins, at least three .beta..sub.2 integrins are known, each comprised of a unique .alpha. subunit in association with a .beta..sub.2 subunit (designated CD18) Kishimoto, et al., Cell 48:681-690 (1987)!. For a recent review of the state of the art with regard to .beta..sub.2 integrins, see Springer, Cell 76:301-314 (1994). CD11a/CD18, also known as .alpha..sub.L .beta..sub.2 or LFA-1, is expressed on all leukocytes and has been shown to bind to ICAM-1, ICAM-2, and ICAM-3. CD11b/CD18, also know as .alpha..sub.M .beta..sub.2 or Mac-1, is expressed on polymorphonuclear neutrophils, monocytes and eosinophils and has been shown to bind to ICAM-1, complement factor iC3b, factor X, and fibrinogen. CD11c/CD18, also known as .alpha..sub.X .beta..sub.2 or p150,95, is expressed on monocytes, polymorphonuclear neutrophils and eosinophils and has been shown to bind to complement factor iC3b and fibrinogen. In addition, a fourth human .beta..sub.2 integrin, designated .alpha..sub.d .beta..sub.2, has recently been identified Van der Vieren, et al., Immunity 3:683-690 (1995)!. Recently, it has been demonstrated that the actin-binding protein, filamin, directly binds to a cytoplasmic portion of .beta..sub.2 subunits Sharma, et al., J. Immunol. 154:3461-3470 (1995)! which suggests a role for one or more of the .beta..sub.2 integrins in formation of focal contacts and cell motility in general see review in Arnaout, Blood 75:1037 (1990)!.
A second subset of leukocyte specific integrins may be referred to as the .alpha..sub.4 integrins in view of the fact that both members of the family are comprised of a common .alpha..sub.4 subunit in association with either a , .beta..sub.1 or .beta..sub.7 subunit. For a recent review, see Springer, supra. VLA-4, also referred to as .alpha..sub.4 .beta..sub.1 or CD49d/CD29, is expressed on most peripheral blood leukocytes except neutrophils and specifically binds VCAM-1 and fibronectin. LPAM-1, also known as .alpha..sub.4 .beta..sub.7 , is expressed on all peripheral blood leukocytes and has been shown to bind MadCAM-1, fibronectin and VCAM-1. Expression of either of the .alpha..sub.4 integrins has also been demonstrated in a wide range of leukocyte cell types in lymphoid organs and in various tissues Hemler et al, Immunol. Rev. 114:45-60, 1990; Kilshaw et al., Eur. J. Immunol 20:2201-2207, 1990; Schweighoffer et al., J. Immunol 151:717-729, 1993; and Lazarovits and Karsh, J. Immunol. 151:6482-6489, 1993). Consistent with the observed participation of .beta..sub.2 integrins in formation of focal contacts, presumably through filamin binding, it has previously been shown that cytoplasmic portions of .beta..sub.1 integrins directly bind .beta.-actinin in vitro. While this interaction has not been demonstrated in vivo, it suggests physiological involvement of .beta..sub.1 integrins in cell mobility and/or maintenance of cell morphology see review in Clark and Brugge, Science 268:233-238 (1995)!.
A number of in vitro and in vivo studies utilizing anti-.alpha..sub.4 monoclonal antibodies have indicated a role for the .alpha..sub.4 integrins in various pathophysiological conditions see review, Lobb and Hemler, J. Clin. Invest. 94:1722-1728 (1994)!. For example, several investigations have provided evidence that .alpha..sub.4 integrins are involved in leukocyte emigration from peripheral blood into regions of inflammation (Weg, et al., J. Exp. Med. 177:561-566, 1992; Winn and Harlan, J. Clin. Invest. 92:1168-1173, 1993). These observations suggest that anti-.alpha..sub.4 antibodies may be capable of ameliorating integrin-associated disease states, and this therapeutic potential has been demonstrated in several animal disease state models. For example, bolus injection of antibodies to .alpha..sub.4 integrins delayed the onset of paralysis in rat and murine experimental allergic encephalomyelitis (Yednock, et al., Nature 356:63-66, 1992; Baron, et al., J. Exp. Med. 177:57-68, 1993). Prophylactic administration of anti-.alpha..sub.4 antibodies reduced ear swelling in murine contact hypersensitivity models (Ferguson, et al., J. Immunol. 150:1172-1182, 1993; Nakajima, et al., J. Exp. Med. 179:1145-1154, 1994). Further, anti-.alpha..sub.4 antibodies were shown to reduce infiltration of pancreatic islets and delay the onset of diabetes in non-obese diabetic mice which are prone to spontaneous development of type I diabetes (Yang, et aL, Proc. Natl. Acad. Sci. (USA) 90:10494-10498. 1993; Burkly, et al., Diabetes 43:529-534, 1994; Baron, et al., J. Clin. Invest. 93:1700-1708, 1994). Still other in vivo studies using anti-.alpha..sub.4 antibodies suggest a role for .alpha..sub.4 integrins in allergic lung inflammation (Pretolani, et al., J. Exp. Med. 180:795-805 (1994); Milne and Piper, Br. J. Pharmacol. 112:82Pa(Abstr), 1994); inflammatory bowel disease (Podolsky, et al., J. Clin. Invest. 92:372-380, 1993); cardiac allograft rejection (Paul, et al, Transplantation 55:1196-1199, 1993); acute nephrotoxic nephritis (Mulligan, et al., J. Clin. Invest. 91:577-587, 1993); and immune complex mediated lung injury (Mulligan, et al., J. Immunol. 159:2407-2417, 1993).
Thus there exists a need in the art to identify molecules which bind to and/or modulate the binding and/or signaling activities of the integrins and to develop methods by which these molecules can be identified. The methods, and the molecules thereby identified, will provide practical means for therapeutic intervention in u integrin-mediated immune and inflammatory responses.